Fusion Gene Product Research Articles

Clinicopathological profile of ependymomas with special reference to survival data - Experiences of a tertiary care center'.

Ependymomas exhibit heterogeneity across age, location, histology, molecular nature and survival suggestive of an epigenetic component in its pathogenesis. The CNS WHO classification (2021) classifies ependymomas based on DNA methylation profiles. Studies suggest that molecular sub-types remain stable throughout the course of disease. Immunohistochemical expression of L1CAM, has been identified as a surrogate marker for ZFTA/c11orf95-RELA fusion in supratentorial ependymomas. This study aims at realising its utility specially in resource-poor setups. Forty-three histopathologically-proven cases of ependymoma under treatment over the period of three years were selected. Histopathological examination followed by routine IHC staining for GFAP, S-100, EMA and Ki-67 in all cases and L1CAM in the supratentorial ependymomas was done. We have followed-up almost all cases during our study period and was correlated with the IHC expression patterns and clinico-pathological parameters, including survival. In our study the commonest location for ependymomas was spine in adults and posterior fossa in pediatric age group. Majority cases belonged to CNS WHO Grade 2 both in adults and in the paediatric age group. Supratentorial location of ependymomas with positive immuno-reactivity for L1CAM and a higher Ki-67 labelling index were associated with poor survival. Our study revealed that L1CAM is an effective surrogate marker for supratentorial ependymomas possibly carrying the ZFTA Fusion gene product. The L1CAM immuno-reactivity also corresponded with the survival data. However, larger population-based studies are required to validate these results further.

Abstract 1681: Characterizing signaling and localization of Daple-FLT3 gene fusion in leukemia cells

Abstract Receptor tyrosine kinases (RTKs) are cell surface receptors that regulate cell survival, cell proliferation, and cell migration upon ligand binding. Genetic mutations leading to hyperactivation of RTKs are often seen in cancer and allow signaling pathways to be constitutively active. In some hematological malignanices, the coiled-coil domain (CCD) of Daple, a cell signaling scaffold protein, has been found in gene fusions with the RTKs FLT3 and PDGFRB, and to the intracellular kinase JAK2. It is hypothesized that this gene fusion product causes the tyrosine kinase domain of FLT3 to become activated and mislocalizes in the cell, leading to rampant cell signaling of either the MAPK, AKT, and STAT5 signal transduction cascades. Using fluorescently tagged Daple-FLT3, it was shown that the gene fusion product is subcellularly localizes to the peri-centrosome space across all leukemia cell lines tested. Ectopic expression demonstrated activation of the kinase domain and subsequent increases in phosphorylation of STAT5α and AKT, but no significant increase in the phosphorylation of MAPK was observed. Further investigation is needed to identify direct substrates of the Daple-FLT3 gene fusion and transcriptional changes. Identifying the direct kinase substrates and insights gained through understanding the gene fusion localization, affects on cell signaling, and changes in gene expression, may unveil novel therapeutic strategies into targeting this protein in cancer. Citation Format: Michael Acquazzino, Jason Ear. Characterizing signaling and localization of Daple-FLT3 gene fusion in leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1681.

A Combination of Alectinib and DNA-Demethylating Agents Synergistically Inhibits Anaplastic-Lymphoma-Kinase-Positive Anaplastic Large-Cell Lymphoma Cell Proliferation.

The recent evolution of molecular targeted therapy has improved clinical outcomes in several human malignancies. The translocation of anaplastic lymphoma kinase (ALK) was originally identified in anaplastic large-cell lymphoma (ALCL) and subsequently in non-small cell lung carcinoma (NSCLC). Since ALK fusion gene products act as a driver of carcinogenesis in both ALCL and NSCLC, several ALK tyrosine kinase inhibitors (TKIs) have been developed. Crizotinib and alectinib are first- and second-generation ALK TKIs, respectively, approved for the treatment of ALK-positive ALCL (ALK+ ALCL) and ALK+ NSCLC. Although most ALK+ NSCLC patients respond to crizotinib and alectinib, they generally relapse after several years of treatment. We previously found that DNA-demethylating agents enhanced the efficacy of ABL TKIs in chronic myeloid leukemia cells. Moreover, aberrant DNA methylation has also been observed in ALCL cells. Thus, to improve the clinical outcomes of ALK+ ALCL therapy, we investigated the synergistic efficacy of the combination of alectinib and the DNA-demethylating agent azacytidine, decitabine, or OR-2100 (an orally bioavailable decitabine derivative). As expected, the combination of alectinib and DNA-demethylating agents synergistically suppressed ALK+ ALCL cell proliferation, concomitant with DNA hypomethylation and a reduction in STAT3 (a downstream target of ALK fusion proteins) phosphorylation. The combination of alectinib and OR-2100 markedly altered gene expression in ALCL cells, including that of genes implicated in apoptotic signaling, which possibly contributed to the synergistic anti-ALCL effects of this drug combination. Therefore, alectinib and OR-2100 combination therapy has the potential to improve the outcomes of patients with ALK+ ALCL.

Abstract 881: Functional characterization and clinical prevalence of ESR1 fusions in advanced endocrine resistant breast cancer

Abstract Breast cancer is the most frequently diagnosed women’s cancer with one in eight women being diagnosed during her lifetime. Even within the era of novel therapeutics and improved diagnostic screening, mortality rates remain stable, with an estimated 43,600 deaths occurring in 2021 alone. Malignant transformation of breast cancer to advanced disease is the mainstay driver of mortality and is largely attributed to treatment resistance. Fortunately, identification of therapeutic resistance mechanisms has rapidly transformed our understanding of cancer evasion and is enabling personalized treatment guidance and regimens. Notably, mutations and amplifications of the estrogen receptor (ER, ESR1) remain a prominent source of endocrine therapy resistance. Two-thirds of all breast cancer are positive for ER, making it an attractive druggable target that has revolutionized patient care. Unfortunately, however, 30% of tumors exposed to endocrine therapy subsequently develop resistance. Recently identified ER fusion proteins, discovered in metastatic endocrine resistant disease, result in substitution of the C-terminal ER ligand binding domain with a variety of fused in-frame gene partners due to genomic translocations occurring at ESR1 intron 6. Our lab has demonstrated that ER fusion proteins drive robust ER hyperactivation and metastatic potential independent of ER ligand estradiol and in the presence of endocrine therapies such as Fulvestrant. Overexpression of ER fusion proteins, particularly ESR1-SOX9 and ESR1-YAP1, in invasive ductal and invasive lobular carcinoma models with endogenous ER knockdown, resulted in enhanced 3D growth, increased migration by wound scratch assay, enhanced colony formation as a proxy of survival and ER pathway hyperactivation. Targeted endogenous ER knockdown utilizing short hairpin RNA enhances fusion driven phenotypes that are unique to fusion partner proteins and cellular context. ER fusion prevalence in advanced breast cancer remains a challenge due to the promiscuous fusion partners disrupting traditional sequencing techniques. Our group has developed a novel method of ESR1 fusion detection, EnRich, which exploits the ESR1 fusion breakpoints. Importantly, we have assessed EnRich fusion discovery with the use of circulating tumor DNA (ctDNA) from non-invasive liquid biopsies. Our pilot study of 15 blood ctDNA samples detected three different ESR1 fusion gene products which have been further validated by PCR. EnRich will help us identify and monitor treatment resistance of known and novel ESR1 fusions in patient samples irrespective of treatment regime as well as in longitudinal samples collected from patients undergoing endocrine therapy. The in vitro resistant properties of fusion proteins emphasize the necessity to better detect these mutations in patients with advanced disease and determine more appropriate treatment measures. Citation Format: Megan E. Yates, Tiantong Liu, Jagmohan Hooda, Jennifer M. Atkinson, Adrian V. Lee, Steffi Oesterreich. Functional characterization and clinical prevalence of ESR1 fusions in advanced endocrine resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 881.

Selection-free endogenous tagging of cell lines by bicistronic co-expression of the surface antigen NGFR

Endogenous protein tagging, in contrast to exogenous overexpression of tagged proteins, allows to characterize specific protein functions under defined physiological or pathophysiological conditions without the influence of non-physiological protein levels. The development of generic and homology-independent tagging strategies, exploiting the CRISPR/spCas9 gene editing system in combination with generic tag donor plasmids, allows targeted and precise gene modification in mammalian cells for almost any desirable gene.So far, fluorescent tags or antibiotic resistance cassettes coupled to the endogenous fusion protein expression have been applied to isolate correctly modified clones. However, both can be challenging, especially when endogenously controlled expression of the tagged protein is weak or regulated by cellular signals. Here, we expand the strategy to selection-free endogenous tagging by exploiting exogenous co-expression of surface antigens. These endogenously regulated, but still easily accessible surface antigens allow simple identification and isolation of clones harboring correctly tagged alleles via common sorting procedures (e.g. FACS/MACS).Using metabolically controlled interaction studies of the endogenously tagged mTORC1-regulating GATOR2 complex protein WDR59, we show that endogenous GFP-labeling does not affect complex association of fusion proteins and downstream signaling via mTORC1. In addition, exogenous co-expression of the NGFR surface antigen does not influence conditional protein-protein interactions.•A method for selection-free, site-specific, homology-independent endogenous genetic tagging.•Production of fusion genes for protein visualization in living cells or determination of protein-protein-interactions.•Expression of a fusion protein mirroring physiological expression in its natural genetic context.

Towards Raman-Based Screening of Acute Lymphoblastic Leukemia-Type B (B-ALL) Subtypes.

Simple SummaryAcute lymphoblastic leukemia (ALL) is the most common pediatric malignancy originating from abnormal lymphoid progenitor cells. Since ALL is genetically highly heterogenous, more sensitive and rapid methods for identifying the molecular subtype of ALL are still being searched, and Raman spectroscopy (RS) has a chance of becoming a valuable tool for this purpose. Herein, the RS was applied to analyze normal B cells and three subtypes of B-ALL, characterized by the presence of the product of gene fusion, i.e., BCR-ABL1, TEL-AML1, and TCF3-PBX1. The classification and discrimination of normal and neoplastic cells were carried out with the chemometric approach. Normal B cells were characterized mostly by bands assigned to nucleic acids and proteins, whereas three subtypes of ALL appeared to contain a higher lipid content. Spectral differences between particular ALL subtypes were modest. The results lead to the conclusion that RS has the potential as a diagnostic tool in clinical practice.Acute lymphoblastic leukemia (ALL) is the most common type of malignant neoplasms in the pediatric population. B-cell precursor ALLs (BCP-ALLs) are derived from the progenitors of B lymphocytes. Traditionally, risk factors stratifying therapy in ALL patients included age at diagnosis, initial leukocytosis, and the response to chemotherapy. Currently, treatment intensity is modified according to the presence of specific gene alterations in the leukemic genome. Raman imaging is a promising diagnostic tool, which enables the molecular characterization of cells and differentiation of subtypes of leukemia in clinical samples. This study aimed to characterize and distinguish cells isolated from the bone marrow of patients suffering from three subtypes of BCP-ALL, defined by gene rearrangements, i.e., BCR-ABL1 (Philadelphia-positive, t(9;22)), TEL-AML1 (t(12;21)) and TCF3-PBX1 (t(1;19)), using single-cell Raman imaging combined with multivariate statistical analysis. Spectra collected from clinical samples were compared with single-cell spectra of B-cells collected from healthy donors, constituting the control group. We demonstrated that Raman spectra of normal B cells strongly differ from spectra of their malignant counterparts, especially in the intensity of bands, which can be assigned to nucleic acids. We also showed that the identification of leukemia subtypes could be automated with the use of chemometric methods. Results prove the clinical suitability of Raman imaging for the identification of spectroscopic markers characterizing leukemia cells.

Comparison of RNA-Based Next-Generation Sequencing Assays for the Detection of NTRK Gene Fusions

Recently, the US Food and Drug Administration approved several targeted therapies directed against oncogenic fusions. One of the most effective such targeted therapies is Vitrakvi (larotrectinib), highly specific oral tropomyosin receptor kinase inhibitor indicated for the treatment of patients with any solid tumor harboring a fusion involving one of the neurotrophic receptor tyrosine kinase (NTRK) genes. Although several diagnostic approaches can be used to detect these NTRK fusions, RNA-based next-generation sequencing remains one of the most sensitive methods, as it can directly detect the transcribed end product of gene fusion at the mRNA level. In this study, performance characteristics of three RNA-based next-generation sequencing assays with distinct mechanisms and chemistries were investigated: anchored multiplex PCR, amplicon-based multiplex PCR, and hybrid capture-based enrichment method. Analytical sensitivity analysis shows that the amplicon-based multiplex PCR method has the lowest limit of detection. However, both hybrid-capture and anchored multiplex PCR methods can detect NTRK fusions with uncommon or novel fusion partners, which is challenging for the amplicon-based multiplex method. As for clinical sensitivity, all three methods were highly concordant in detecting NTRK fusions in patient samples. Additionally, they all presented equivalent high-level performance in specificity, suggesting that all three platforms can detect NTRK fusions in clinical samples with similar performance characteristics.

OS06.4A Identification of T cell receptors targeting ZFTA-RELA fusion-positive ependymoma

Abstract BACKGROUND The oncogenic gene fusion between ZFTA (formerly C11orf95) and RELA has in recent years been highlighted as oncogenic driver event ultimately leading to malignant transformation and progression of ependymoma. Representing a genetic hallmark in 17.6% of ependymomas, ZFTA-RELA fusion-positive tumors qualified as separate diagnostic entity in the 2016 revision of the WHO classification of CNS tumors. This tumor entity mainly occurs in pediatric patients and is characterized by poor prognosis and the lack of biology-driven therapy. RESULTS De novo prediction of putative gene fusion-derived neoepitopes in malignant CNS diseases yielded several potential candidates suitable for screening. Of these, vaccination of A2.DR1-transgenic major histocompatibility complex (MHC)-humanized mice with in silico compiled peptide vaccines encompassing the ZFTA-RELA fusion sequence elicited robust antigen-specific CD4+-restricted immune responses. We identified ZFTA-RELA-reactive T cell clones by multiplexed Interferon-γ / Interleukin-2 recall response. Single-cell VDJ-sequencing of reactive T cells demonstrated a highly clonal T cell receptor (TCR) repertoire and shared clonotypes among all vaccinated animals. Matching TCRα/β chains have been assembled from single-cell sequencing data and cloned for functional testing of neoepitope-reactive TCR-transgenic T cells in vitro as well as in vivo using a transposon system-based immunocompetent, MHC-humanized ZFTA-RELA fusion-positive ependymoma model. CONCLUSION Here we identify the oncogenic ZFTA-RELA gene fusion product as a novel tumor-specific neoepitope that is recognized by MHC class II-restricted TCRs. Therapeutic efficacy of TCR-transgenic cell therapy can be further investigated using an immunocompetent MHC-humanized mouse model of ZFTA-RELA fusion-positive ependymoma. Our findings provide initial evidence for neoepitope-specific immunotherapy in pediatric CNS tumors.

Current Trends in Prevalence and Role of Long Noncoding RNA and Gene Fusion in Prostate Cancer: An Overview

Objectives The aim of this study is to analyze the current scenario in the diagnostic modalities for prostate cancer. Materials and Methods We searched PubMed, Google Scholar, and ResearchGate for relevant data. Articles published in the last 10 years were taken into consideration. The role of long noncoding RNA and gene fusion products in the context of prostate cancer was reviewed, which included their roles in diagnosis, prognosis, and assessment of response to therapy. Results Several long noncoding RNAs (lncRNA) have been isolated and have been shown to be useful in diagnosing and prognosticating prostate cancer. We have also looked into the role of TMPRSS2:ERG gene fusion in prostate carcinoma diagnosis. These molecular parameters have been looked into due to the fact that the current parameters in use such as prostate-specific antigen have several drawbacks that limit their potential.

Potential role of chimeric genes in pathway-related gene co-expression modules

BackgroundGene fusion has epigenetic modification functions. The novel proteins encoded by gene fusion products play a role in cancer development. Therefore, a better understanding of the novel protein products may provide insights into the pathogenesis of tumors. However, the characteristics of chimeric genes are rarely studied. Here, we used weighted co-expression network analysis to investigate the biological roles and underlying mechanisms of chimeric genes.MethodsDownload the pig transcriptome data, we screened chimeric genes and parental genes from 688 sequences and 153 samples, predict their domains, and analyze their associations. We constructed a co-expression network of chimeric genes in pigs and conducted Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis on the generated modules using DAVID to identify key networks and modules related to chimeric genes.ResultsOur findings showed that most of the protein domains of chimeric genes were derived from fused pre-genes. Chimeric genes were enriched in modules involved in the negative regulation of cell proliferation and protein localization to centrosomes. In addition, the chimeric genes were related to the growth factor-β superfamily, which regulates cell growth and differentiation. Furthermore, in helper T cells, chimeric genes regulate the specific recognition of T cell receptors, implying that chimeric genes play a key role in the regulation pathway of T cells. Chimeric genes can produce new domains, and some chimeric genes are a key role involved in pathway-related function.ConclusionsMost chimeric genes show binding activity. Domains of chimeric genes are derived from several combinations of parent genes. Chimeric genes play a key role in the regulation of several cellular pathways. Our findings may provide new directions to explore the roles of chimeric genes in tumors.

Abstract 4223: Combination therapy of BCR-ABL-positive B cell acute lymphoblastic leukemia by tyrosine kinase inhibitor dasatinib and c-Jun N-terminal kinase inhibition

Abstract The Philadelphia chromosome (Ph), which leads to creation and expression of the fusion gene product BCR-ABL, underlines the pathogenesis of chronic myelogenous leukemia (CML) and a fraction of adult and pediatric acute B-lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitors (TKIs) has shown a remarkable clinical activity in patients with CML, but their efficacy in treating Ph+ B-ALL is limited. Identifying additional therapeutic targets is important for the effective treatment of Ph+ B-ALL. In this study, we found that the c-Jun N-terminal kinase (JNK) signaling pathway is abnormally activated in both human and mouse BCR-ABL+ B-ALL cells, but the BCR-ABL TKI does not inhibit JNK activation in these cells. Inhibition of JNK, either by RNAi-mediated downregulation or by JNK inhibitors, could significantly reduce viability of Ph+ B-ALL cells. JNK inhibition by RNAi-mediated downregulation or JNK inhibitors also showed a synergistic effect with the BCR-ABL TKI, dasatinib, in killing Ph+ B-ALL cells in vitro. Furthermore, a potent JNK inhibitor, JNK-IN-8, in combination with dasatinib markedly improved the survival of mice with BCR-ABL induced B-ALL, as compared to the treatment with dasatinib alone. Our findings indicate that simultaneously targeting both BCR-ABL and JNK kinase might serve as a promising therapeutic strategy for Ph+ B-ALL. Citation Format: Xinhua Xiao, Bo Jiao, Ruibao Ren. Combination therapy of BCR-ABL-positive B cell acute lymphoblastic leukemia by tyrosine kinase inhibitor dasatinib and c-Jun N-terminal kinase inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4223.

Combination therapy of BCR-ABL-positive B cell acute lymphoblastic leukemia by tyrosine kinase inhibitor dasatinib and c-JUN N-terminal kinase inhibition

BackgroundThe Philadelphia chromosome (Ph), which leads to the creation and expression of the fusion gene product BCR-ABL, underlines the pathogenesis of chronic myelogenous leukemia (CML) and a fraction of adult and pediatric acute B-lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitors (TKIs) have shown a remarkable clinical activity in patients with CML, but their efficacy in treating Ph+ B-ALL is limited. Identifying additional therapeutic targets is important for the effective treatment of Ph+ B-ALL.MethodsActivation of the JNK signaling pathway in human and mouse BCR-ABL+ B-ALL cells with or without dasatinib treatment was analyzed by Western blotting. JNK was inhibited either by RNA interference or chemical inhibitors, such as JNK-IN-8. The effect of JNK inhibition with or without BCR-ABL TKI dasatinib on BCR-ABL+ B-ALL cells was analyzed by the CellTiter-Glo® Luminescent Cell Viability Assay. The in vivo effects of JNK-IN-8 and dasatinib alone or in combination were tested using a BCR-ABL induced B-ALL mouse model.ResultsWe found that the c-JUN N-terminal kinase (JNK) signaling pathway is abnormally activated in both human and mouse BCR-ABL+ B-ALL cells, but the BCR-ABL TKI does not inhibit JNK activation in these cells. Inhibition of JNK, either by RNAi-mediated downregulation or by JNK inhibitors, could significantly reduce viability of Ph+ B-ALL cells. JNK inhibition by RNAi-mediated downregulation or JNK inhibitors also showed a synergistic effect with the BCR-ABL TKI, dasatinib, in killing Ph+ B-ALL cells in vitro. Furthermore, a potent JNK inhibitor, JNK-IN-8, in combination with dasatinib markedly improved the survival of mice with BCR-ABL induced B-ALL, as compared to the treatment with dasatinib alone.ConclusionsOur findings indicate that simultaneously targeting both BCR-ABL and JNK kinase might serve as a promising therapeutic strategy for Ph+ B-ALL.

Comprehensive chromosomal aberrations in a case of a patient with TCF3-HLF-positive BCP-ALL

BackgroundThe use of high-throughput analytical techniques has enabled the description of acute lymphoblastic leukaemia (ALL) subtypes. The TCF3-HLF translocation is a very rare rearrangement in ALL that is associated with an extremely poor prognosis. The TCF3-HLF fusion gene in the described case resulted in the fusion of the homeobox-related gene of TCF3 to the leucine zipper domain of HLF. The TCF3-HLF fusion gene product acts as a transcriptional factor leading to the dedifferentiation of mature B lymphocytes into an immature state (lymphoid stem cells). This process initiates the formation of pre-leukaemic cells. Due to the rarity of this chromosomal aberration, only a few cases have been described in the literature. The advantage of this work is the presentation of an interesting case of clonal evolution of cancer cells and the cumulative implications (diagnostic and prognostic) of the patient’s genetic alterations.Case presentationThis work presents a patient with diagnosed with TCF3-HLF-positive ALL. Moreover, the additional genetic alterations, which play a key role in the pathogenesis of ALL, were detected in this patient: deletion of a fragment from the long arm of chromosome 13 (13q12.2-q21.1) containing the RB1 gene, intragenic deletions within the PAX5 gene and NOTCH1 intragenic duplication.ConclusionsA patient with coexistence of chromosomal alterations and the TCF3-HLF fusion has not yet been described. Identifying all these chromosomal aberrations at the time of diagnosis could be sufficient to determine the cumulative effects of the described deletions on the activity of other oncogenes or tumour suppressors, as well as on the clinical course of the disease. On the other hand, complex changes in the patient’s karyotype and clonal evolution of cancer cells call into question the effectiveness of experimental therapy.

59. Dedifferentiated solitary fibrous tumour arising from the bladder: A case report

Background: Solitary fibrous tumour (SFT) is a rare mesenchymal tumour of fibroblastic differentiation. They most commonly arise in the pleura, but have a wide anatomical distribution. Conventional SFTs are circumscribed, spindle cell neoplasms with characteristic histomorphology including patternless architecture, dense collagen and prominent haemangiopericytoma-like ‘staghorn’ vessels. Conventional SFTs express CD34, Bcl2 and STAT6, but are negative for S100, keratin and desmin. Molecular profiling has shown consistent 12q chromosomal abnormality resulting in NAB2-STAT6 fusion. The NAB2-STAT6 gene fusion product is reliably identified on STAT6 immunohistochemistry, which has proven both a highly sensitive and specific marker for SFTs. Most SFTs are clinically benign, however up to 15% may show features of malignancy including increased mitoses (>4 per 10 HPF), necrosis and infiltrative margins. In addition, areas of dedifferentiation may occur, characterised by sharply demarcated areas of hypercellularity and pleomorphism with loss of morphological features of conventional SFT. Dedifferentiated areas may lose the typical immunohistochemical profile of conventional SFT, including loss of CD34 and STAT6. Aim: To report a case of dedifferentiated SFT in the bladder and review the current literature. Method: A 39-year-old male presented with one month history of urinary frequency and abdominal pain. CT and MRI showed a large heterogenous necrotic mass in the posterior bladder wall. An en bloc tumour resection and cystoprostatectomy was performed. Results: Macroscopic examination showed a 15 cm circumscribed tumour arising from the superior bladder. Microscopy revealed a spindle cell tumour arising from the bladder muscularis propria with areas of conventional SFT adjacent to vast areas of dedifferentiation with malignant features including hypercellularity, pleomorphism, increased mitoses (40 per 10 HPF) and necrosis. Immunohistochemistry showed positive staining for STAT6, CD34 and Bcl2 in both conventional and dedifferentiated areas. Conclusion: This is a unique case of a dedifferentiated SFT of the bladder in a young patient.

Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data.

Versatile Regulator or Integrator RNA-binding Protein TLS/FUS Works for Phase Separation on Regulation of the Human Genome - In Relation to Liposarcoma and ALS

This review article aims at comprehensive understanding of biological functions of RNA-binding proteins (RBPs) and cognate RNAs. For showing the divergent and dispersed aspects of RBP functions of the human genome in living cells, RBP TLS/FUS is chosen for these topics. Recent discovery of TLS as a phase transition or phase separation inducer presents previously unprecedented models regarding phase separation and enhancer functions in the genome. Trends in research altered up to interests or desire of scientists. Research activities of TLS/FUS have been dramatically drifted across over the last century. TLS was identified as a fusion gene product of TLS-CHOP of liposarcoma in 1993. Before announcement of the human genome draft, there were compelling desires to hunt novel genes. Gene cloning from libraries was one of prevalent activities at that time. Next wave came with describing TLS as a causative gene for familial amyotrophic lateral sclerosis (ALS). Investigation of lethal neurodegenerative disease, ALS has social impact. Elucidation of onset of ALS has progressed rapidly. Latest discovery with TLS is its solute for phase separation and phase transition into aggregation. The phase separation and resultant aggregation are pointed out in relation to the onset of ALS. Then, people rush to search for tide of TLS to phase separation in link to cause for ALS. Analysis of the phase separation could provide a novel outline for long distant regulation of gene in the genome. In this review article, the author focuses emerging trendy targets of a versatile molecule TLS and discuss scientific needs behind these orientation for a specific molecule in biological sciences.

Optimization of overlap extension PCR for efficient transgene construction

PCR is a powerful tool for generating specific fragments of DNA that can be used to create gene variations or tagged expression constructs. Overlap extension PCR is a valuable technique that is commonly used for cloning large complex fragments, making edits to cloned genes or fusing two gene elements together. After difficulties in utilizing this technique following existing methods, we developed an optimized protocol. To accomplish this, three significant changes were made; 1) touchdown PCR cycling parameters were used to eliminate the need for optimizing PCR cycling conditions, 2) the high-fidelity, high-processivity Q5 DNA polymerase was used to improve full-length amplification quality, and 3) a reduced amount of primer in the final PCR amplification step decreased non-specific amplimers. This modified protocol results in consistent generation of gene fusion products, with little to no background and enhanced efficiency of the transgene construction process.

Functional heritage: the evolution of chimeric RNA into a gene

ABSTRACT Once believed to be unique features of neoplasia, chimeric RNAs are now being discovered in normal physiology. We speculated that some chimeric RNAs may be functional precursors of genes, and that forming chimeric RNA at the transcriptional level may be a ‘trial’ mechanism before the functional element is fixed into the genome. Supporting this idea, we identified a chimeric RNA, HNRNPA1L2-SUGT1 (H-S), whose sequence is highly similar to that of a ‘pseudogene’ MRPS31P5. Sequence analysis revealed that MRPS31P5 transcript is more similar to H-S chimeric RNA than its ‘parent’ gene, MRPS31. Evolutionarily, H-S precedes MRPS31P5, as it can be detected bioinformatically and experimentally in marmosets, which do not yet possess MRPS31P5 in their genome. Conversely, H-S is minimally expressed in humans, while instead, MRPS31P5 is abundantly expressed. Silencing H-S in marmoset cells resulted in similar phenotype as silencing MRPS31P5 in human cells. In addition, whole transcriptome analysis and candidate downstream target validation revealed common signalling pathways shared by the two transcripts. Interestingly, H-S failed to rescue the phenotype caused by silencing MPRS31P5 in human and rhesus cells, whereas MRPS31P5 can at least partially rescue the phenotype caused by silencing H-S in marmoset cells, suggesting that MRPS31P5 may have further evolved into a distinct entity. Thus, multiple lines of evidence support that MRPS31P5 is not truly a pseudogene of MRPS31, but a likely functional descendent of H-S chimera. Instead being a gene fusion product, H-S is a product of cis-splicing between adjacent genes, while MRPS31P5 is likely produced by genome rearrangement.

Abstract 406: Profiling plasma cell-free RNA (cfRNA) with the NanoString low input nCounter assay

Abstract Background: Liquid biopsy offers advantages over tissue biopsy for early disease diagnosis, monitoring of disease progression, and evaluation of therapeutic interventions because sample collection is rapid, easy, minimally invasive, and repeatable. In particular, measuring cell-free RNA (cfRNA) in the plasma offers the opportunity for direct monitoring of a tumor cell byproduct without the need for direct access to the tumor. However, robust cfRNA detection has been difficult historically due to weak and highly variable signal detection. The nCounter platform and low input profiling protocol have been adapted to enable robust and reproducible detection of cfRNA from plasma, which is demonstrated here in proof of concept experiments using synthetic targets and patient samples to quantify transcription of standard gene transcripts and fusion products. Methods: The standard nCounter detection platform which leverages direct hybridization of optical barcodes to RNA transcripts of interest has been adapted to enable detection of rare targets by leveraging an initial targeted amplification step to increase transcript abundance with minimal bias of relative transcript abundance. Feasibility of this process to detect cfRNA was assessed with synthetic DNA oligos and commercially sourced patient-derived plasma samples. Plasma cfRNA was isolated and concentrated using commercial cfRNA kits. Relative expression of targets was benchmarked to universal reference RNA and compared between samples by both nCounter and qPCR. Results: Using synthetic ultramer DNA oligo targets for 7 genes, the limit of detection of the assay was as low as 24 molecules per target with 100% specificity. Using cfRNA from healthy donors, the nCounter assay was shown to profile relative abundance of 40 targets. 9 genes were selected to benchmark nCounter against qPCR and showed high correlation (R2 = 0.84) with linearity (slope = 0.97). cfRNA from healthy donors or patients with advanced CRC or NSCLC was profiled by the 770-gene PanCancer IO360 panel with a pre-amplification by the adapted nanoString low RNA input protocol. Unsupervised hierarchical clustering revealed that CRC derived cfRNA had different transcriptional profiles from healthy donors, and NSCLC cfRNA had an intermediate profile. Furthermore, a number of therapeutically relevant targets, including EGF, EGFR, PD-1, and PD-L1 could be detected in cfRNA from cancer patients but not in cfRNA from healthy donors. Conclusion: The nCounter low input protocol can be used for transcriptional profiling of cfRNA from healthy and diseased patients with high sensitivity and specificity. The flexible primer design strategy enables profiling of a variety of transcripts, including fusion gene products, with the possibility of profiling up to 800 transcripts from a single sample. In summary, the nCounter platform (RUO) provides a robust flexible molecular profiling tool for liquid biopsy research. Citation Format: Chung-Ying (Alan) Huang, Sarah Warren, Jason Reeves, Megan Wurden, Tim Riordan, Rich Boykin, Mingdong Liu, David Henderson, Dan Hanson, Jay Gerlach, Joseph M. Beechem. Profiling plasma cell-free RNA (cfRNA) with the NanoString low input nCounter assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 406.

Abstract 5133: Identification of distinct gene expression profiles in African Americans and Caucasian men with prostate cancer

Abstract Introduction African Americans (AA) are disproportionately affected by prostate cancer (PCa) compared to European Americans (EA). Genetic and epigenetic differences have been implicated in this racial disparity. Present PCa diagnostic and treatment regimens have focused mainly on gene fusion product involving the androgen driven transmembrane protease serine 2 (TMPRSS2) and the transcription factor, ETS related gene (ERG). However, TMPRSS2:ERG (T2E) fusion product is common mainly to EA (50-80% of patients) and has lower prevalence amongst other ethnicities including AAs (10-30%), Japanese and Chinese patients. In addition, T2E negative PCa subtype has been associated with increased risk of developing castration resistant PCa (CRPC). This study aims at classifying PCa by molecular and pathologic subtypes that drive disease aggressiveness in patients of AA and EA ancestry. Methodology Prostate cancer tissue slides obtained from EA and AA patients were stained for ERG expression. Laser-capture micro-dissected RNAs from formalin-fixed paraffin-embedded tissues were used for RNA-seq. DESeq2 was used as a statistical procedure to call differentially expressed genes (DEGs) in different samples. Data set from Powell was also analyzed for DEGs between ERG positive and ERG negative samples. Leading-Edge Analysis (GSEA v2.2.4) was performed to determine the representation of DEGs across enriched gene sets. Results 32.4% of EA were ERG positive compared to 29% of AA that stained positive. More EA samples (26.4%) had high intensity staining (2≥ on a 1 - 3 scale) compared to AA samples (25.8%). Over 11% of EA samples had high (4) distribution of ERG positive staining compared 6.5% in AA samples. Analysis of the Powell dataset (GSE41696) revealed 301 DEGs and gene set enrichment analysis identified immune system process, regulation of cellular component and homeostatic process as most biological processes affected by DEGs. Other processes significantly affected included: cell motility, cell activation and regulation of locomotion. At least 15 genes including: CCL5, TLR4, PIK3CB, CD86, KIT, CD40, ITGB3, PDGFRA, LEF1, HIF1A, CLU, PLA2G6, GLI3, PIK3CA and TNNB1, were present in at least 10 enriched gene sets. The DEGs across enriched gene sets showed gene signatures involved in Wnt signaling, PI3K, chemokines and receptor tyrosine kinases. Conclusion Specific sets of genes are differentially enriched between EGR-/AR- and ERG+/AR+. These findings suggest that PCas have a distinct gene expression profile in the absence of the T2E fusion, which may act as a driver of disease aggressiveness and racial health disparities in PCa. Identifying these differences in gene-signature expression and signaling transduction pathways will allow stratification of men across races and identify, early on, those individuals who are likely to develop the most aggressive disease. Citation Format: Joakin Mori, Raven Williams, Hui-Xian Lin, Alahni Becks, Jason White, Balasubramanyam Karanam, Clayton Yates, Honghe Wang. Identification of distinct gene expression profiles in African Americans and Caucasian men with prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5133.